Method for detection of mers-cov with igy antibodies

ABSTRACT

A method for detecting MERS-CoV at high sensitivity and specificity using IgY antibodies that bind to MERS-CoV N protein, its fragments and domains. Isolated or purified IgY monospecific antibodies to MERS-CoV N protein.

REFERENCE TO A SEQUENCE LISTING

In accordance with 37 CFR § 1.52(e)(5), the present specification makesreference to a Sequence Listing (submitted electronically as a .txt filenamed “530967US_ST25.txt”. The .txt file was generated on Jan. 25, 2021and is 6.83 kb in size. The entire contents of the Sequence Listing arehereby incorporated by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The invention pertains to the fields of virology, and immunodiagnosticproducts and methods, more specifically, to the use of avian IgYantibodies to detect MERS-CoV nucleocapsid protein (“N protein”)epitopes with high specificity and sensitivity.

Description of Related Art

Middle-East respiratory syndrome coronavirus (MERS-CoV) is an emergingzoonotic virus that causes severe respiratory illness in humans. MERSfirst emerged in Saudi Arabia in 2012 causing an epidemic in the MiddleEast. About 2,500 cases have been reported as of January 2020; worldwideweb.emro.who.int/health-topics/mers-cov/mers-outbreaks.html. RetrievedJan. 27, 2021. About 35% of those who are diagnosed with the disease diefrom it; worldwide web .who.int/news-room/fact-sheets/detail/middle-east-respiratory-syndrome-coronavirus-(mers-cov).Retrieved Jan. 27, 2021. Larger outbreaks have occurred in South Koreain 2015 and in Saudi Arabia in 2018. The World Health Organization (WHO)reports that there have been 2519 confirmed cases with a total of 866deaths in 26 countries including the United States, and that thefatality rate is 34.3%; MERS situation update, January 2020. WorldHealth Organization. January 2020. Respiratory infections by MERS-CoVrepresent a global threat that affects millions of people, especiallythe immunocompromised, children, and the elderly.

Dromedary camels represent an animal reservoir for MERS-CoV from whichzoonosis can occur. The vast majority of dromedaries in the ArabianPeninsula are reported to be seropositive for MERS-CoV and MERS-CoVstrains found in epidemiologically-linked humans and dromedary camelsare almost identical; Azhar, E. I., et al., Evidence for camel-to-humantransmission of MERS coronavirus. NEW ENGLAND JOURNAL OF MEDICINE, 2014,370(26): 2499-2505. However, small outbreaks from countries outsideSaudi Arabia such as the UK, Europe, USA, and China have providedevidence of human-to-human transmission.

Currently, there are no clinically approved treatments or vaccinesagainst MERS-CoV. Thus, rapid laboratory identification of the infectedanimals and humans and contact tracing is critical in controlling spreadof the infection.

RT-RCR is the current method of choice for the diagnosis of MERS-CoV andhas high sensitivity and specificity; however, nucleic acid-based testsrequire molecular skills and specialized equipment making themunsuitable as point-of-care testing (POCT) or for rapid bedsidediagnosis.

An alternative to RT-RCR is antigen- or antibody-based immunodiagnostictesting. However, existing immunodiagnostic tests for MERS-CoV have anumber of limitations.

Antibodies to MERS-CoV usually require immunization of mammals toproduce either monoclonal or polyclonal antibodies, followed by repeatedinvasive blood collection from the immunized mammal, and subsequenteuthanasia of the mammal.

For human immunodiagnostics, mammalian antibodies often cross-react orare subject to interference by other mammalian proteins contained in adiagnostic sample. For example, mammalian antibodies can react withrheumatoid factor (RF), mammalian complement components, or mammalian Fcreceptors. Moreover, monoclonal antibodies which are typically producedin mice can interact with human anti-mouse IgG antibodies (HAMA). Theseinteractions make immunodiagnostic testing less accurate due to falsepositive results caused by these interactions.

Immunodiagnostic tests which use antibodies that recognize MERS-CoV Sprotein (a surface exposed antigen) can lack specificity due tovariation of the amino acid sequences of S proteins. Such variationleads to S proteins from different strains of MERS-CoV presentingdifferent epitopes. This can produce false negative results or lowersensitivity because antibodies to S protein from one strain of MERS-CoVmay not recognize S protein epitopes from a different strain or onlyrecognize a subset of such epitopes leading to lower sensitivity.

Moreover, the immunological repertoires of mammals may not produceantibodies that recognize important epitopes of MERS-CoV, for example,segments of N protein that resemble conserved mammalian (or “self”)proteins may not be recognized by the mammalian immune system.Additionally, mammalian antibody repertoires may constitute too fewsub-types to recognize certain diagnostic or therapeutic epitopes ofMERS-CoV or may contain antigen processing and presentation systems thatfail to present certain MERS-CoV epitopes.

In view of these challenges to existing detection methods, the inventorssought to develop highly specific, sensitive, economical, rapid, anduser friendly methods for detection of MERS-CoV for use in thediagnostic laboratory as well as in the field or at the bedside.

The inventors considered that differences in genetic background andphylogenetic distance between avians and mammals as well as indifferences in coronavirus tropism might be reflected by better orbroader IgY antibody responses to MERs-CoV N protein in comparison toantibody responses produced by mammals. Such a broader or betterresponse may be reflected by differences in the numbers of N proteinepitopes recognized by avian IgY or by higher binding affinity of IgYantibodies to particular epitopes of N protein.

The inventors also recognized that immunoassays that use homologousmammalian antibodies may negatively impact assay performance byproducing false negative or false positive readings. For example,immunoassays using mammalian immunoglobulins as bioactive molecules tocapture or detect the analyte are affected by heterophilic antibodiesand/or high levels of non-specific antibody binding. As mentioned above,nonspecific binding can occur between mammalian anti-MERS-CoV antibodiesand other components of the mammalian immune system or hematologicalsystem. For example, mammalian antibodies specific for MERS-CoV can withrheumatoid factor (RF), complement components or mammalian Fc receptors,thus producing false-positive results. False positives may also resultfrom the presence of heterophilic human antibodies, or human antibodiesthat bind to animal antibodies used in an immunochemistry assay, such asmurine antibodies. False negative readings can be produced by failure ofan antibody to recognize epitopes of an analyte (like a MERS-CoVantigen) or lack of sufficient binding affinity for epitopes of theanalyte; or by interference by serum proteins with recognition of theanalyte by an analyte-specific antibody.

BRIEF SUMMARY OF THE INVENTION

In view of these problems, the inventors sought to determine whether IgYantibodies from chickens, either as polyclonal or monoclonalpreparations, might offer recognizable advantages over their mammalianhomologues for immunodiagnosis of MERS-CoV. With these potentialadvantages in mind, the inventors sought to investigate, develop andcharacterize IgY antibodies to MERS-CoV N protein which could provide abroader or different ability to recognize MERS-CoV epitopes than theirmammalian homologues. They also sought to identify IgY antibodies havinga higher binding affinity or avidity for MERS-CoV N protein thanmammalian homologues of IgY like IgG or IgE antibodies.

Embodiments of the invention include, but are not limited to thefollowing.

One embodiment of this technology is an assay for detecting MERS-CoV ina sample comprising contacting the sample with an IgY antibody thatrecognizes MERS-CoV N protein (“NP”) and detecting complex formation orbinding of the IgY to the MERS-CoV. Preferably, the IgY antibody thatrecognizes N protein is a polyclonal antibody or a polyclonalmonospecific antibody to a particular epitope or segment of N protein,for example, a peptide segment or fragment of the N protein sequencethat comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 or >20 contiguous amino acid residues. In otherembodiments, the monospecific antibody may specifically bind to longersegments of N protein such as to the N or C domains of N protein. Inother embodiments, the IgY antibody may be monoclonal antibody ormixture of 2, 3, 4, 5, 6 or more monoclonal antibodies that bind to Nprotein. It may also be polyclonal serum derived from an immunizedchicken or other avian that has not been further purified to removeantibodies that do not bind to N protein.

This assay may be a direct or indirect ELISA assay, such as an ELISAsandwich assay, wherein the IgY antibody that recognizes MERS-CoV NP isa capturing antibody bound to a substrate, wherein the capturingantibody bound to the substrate is contacted with a sample suspected ofcontaining MERS-CoV, and wherein complex formation is measured by thebinding of a tagged secondary antibody that recognizes MERS-CoV.

It may also constitute an immunoswab assay, which comprises contacting asample with IgY antibodies bound to a swab (such as a swab comprisingcalcium alginate), removing unbound sample, and detecting complexformation. Swabs suitable for use as immunoswabs are commerciallyavailable.

The IgY-based assays disclosed herein may detect N protein in abiological sample containing no more than 10, 15, 20, 25, 30, 35, 40, 45or 50 ng/ml of N protein.

Preferably, the IgY antibody does not substantially interact withrheumatoid factor (RF), human anti-mouse IgG antibodies (HAMA), or otherheterophilic antibodies, mammalian complement components or otherserological proteins, and/or mammalian Fc receptors.

In the assays disclosed herein the IgY antibody is a polyclonalantibody, monospecific antibody which may be polyclonal, or monoclonalantibody. In some embodiments, the polyclonal antibody is produced byimmunizing chickens with isolated or recombinant NP and recovering IgYantibody from eggs laid by the immunized chickens.

Monospecific antibodies to particular determinants or epitopes of Nprotein may be isolated by affinity purification using such N proteindeterminants. Monospecific antibodies may also be obtained by immunizinga chicken or other avian with a particular peptide fragment of Nprotein, for example, a chicken may be immunized with the N or C domainof N protein so that only antibodies to the N or C domain are induced.

In some embodiments of the assay, the IgY antibody binds to at least oneepitope within the amino acid sequence described by SEQ ID NO: 1 or an Nprotein analog having at least 95, 96, 97, 98, 99, 99.5, 99.9% or moresimilarity or identity thereto or to an N protein having up to 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23,24, or 25 amino acid insertions, substitutions and/or deletions to SEQID NO: 1. A nucleoprotein comprising SEQ ID NO: 1 is available from SinoBiological and was used for development of the monospecific anti-NP IgYantibodies disclosed herein. This NP sequence and associated structuralor functional features are incorporated by reference to GenBank:AFS88943.1.

BLASTP can be used to identify an amino acid sequence having at least70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99%, 99.5%,or >99.5% sequence identity or similarity to a reference amino acidusing a similarity matrix such as BLOSUM45, BLOSUM62 or BLOSUM80 whereBLOSUM45 can be used for closely related sequences, BLOSUM62 formidrange sequences, and BLOSUM80 for more distantly related sequences.Unless otherwise indicated a similarity score will be based on use ofBLOSUM62. When BLASTP is used, the percent similarity is based on theBLASTP positives score and the percent sequence identity is based on theBLASTP identities score. BLASTP “Identities” shows the number andfraction of total residues in the high scoring sequence pairs which areidentical; and BLASTP “Positives” shows the number and fraction ofresidues for which the alignment scores have positive values and whichare similar to each other. Amino acid sequences having these degrees ofidentity or similarity or any intermediate degree of identity orsimilarity to the amino acid sequences disclosed herein are contemplatedand encompassed by this disclosure. A representative BLASTP setting usesan Expect Threshold of 10, a Word Size of 3, BLOSUM 62 as a matrix, andGap Penalty of 11 (Existence) and 1 (Extension) and a conditionalcompositional score matrix adjustment. Default settings for BLASTP aredescribed by and incorporated by reference to hypertext transferprotocol://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LI NK_LOC=blasthome (lastaccessed Oct. 2, 2020).

In one embodiment, the IgY antibody comprises a monospecific antibodythat binds to a peptide epitope of N protein that ranges in length from6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 to 20 amino acids ofthe amino acid sequence described by SEQ ID NO: 1 or an analog thereof.

In another embodiment, the IgY antibody comprises a monospecificantibody that binds to an N-terminal domain segment comprising residues1-36 of SEQ ID NO: 1.

In another embodiment, the IgY antibody comprises a monospecificantibody that binds to an N-terminal domain of MERS CoV N proteincomprising residues 37-164 of SEQ ID NO: 1.

In another embodiment the IgY antibody comprises a monospecific antibodythat binds to a central segment of MERS CoV N protein comprisingresidues 165-238 of SEQ ID NO: 1; or that binds to a disordered regionor LKR containing a stretch of serine and arginine residues andcomprising residues 156 to 206 of SEQ ID NO: 1 or

(SEQ ID NO: 3) LPKNFHIEGTGGNSQSSSRASSVSRNSSRSSSQGSRSGNSTRGTSPGPSG I.

In another embodiment, the IgY antibody comprises a monospecificantibody that binds to a C-terminal domain of MERS CoV N proteincomprising residues 239-362 of SEQ ID NO: 1.

In another embodiment, the IgY antibody comprises a monospecificantibody that binds to a C-terminal segment of MERS CoV N proteincomprising residues 363-413 of SEQ ID NO: 1.

In some embodiments, the IgY antibody comprises a monospecific antibodythat binds to a peptide comprising positions 4-12, 42-47, 50-58, 60-66,92-102, 108-126, 144-150, 172-180, 206-225, 230-240, 261-268, 296-304,324-337, 347-353 or 399-405 described by SEQ ID NO: 1. These segments ofN protein have been identified using bioinformatics as B cell epitopes.These and the description of humoral and cellular epitopes anddeterminants of N protein and bioinformatic methods for identifying themare incorporated by reference to Jiandong Shi, et al. Epitope-BasedVaccine Target Screening against Highly Pathogenic MERS-CoV: An InSilico Approach Applied to Emerging Infectious Diseases, PLOS ONE,December 2015, hypertext transfer protocolsecure://doi.org/10.1371/journal.pone.0144475. The correspondingepitopes and N protein sequences as given by the above reference arealso incorporated by reference.

In an alternative embodiment, any of the B cell epitopes herein may bemodified by 1 or 2 deletions, substitutions, or insertions of an aminoacid. Such epitopes may also be conjugated to other moieties or embeddedin a longer endogenous or exogenous carrier protein to improve theirimmunogenicity or their ability to bind to IgY antibodies.

In other embodiments, the IgY antibody comprises a monospecific antibodythat binds to a peptide segment consisting of the following residues ofa polypeptide comprising SEQ ID NO: 1:

K346, W347, L348, and E349;

Y327, F328, L329, and R330;

L350, L351, E352, 0353, N354, 1355, D356, A357, Y358, K359, T360, F361,P362, K363, K364, and E365;

D320, D321, H322, G323, N324, P325, and V326;

H252, K253, R254, T255, 5256, T257, K258, 5259, F260, N261, M262, V263,Q264, A265, F266, G267, L268, R269, G270, P271, G272, D273, L274, Q275,G276, N277, F278, G279, D280, L281, Q282, L283, N284, K285, L286, G287,T288, E289, D290, P291, and R292;

R399, P400, S401, V402, Q403, P404, G405, P406, M407, I408, D409, V410,N411, T412, and D413;

W293, P294, 0295, 1296, A297, and E298;

F312, K313, L314, T315, H316, Q317, N318, and N319; and

L299, A300, P301, T302, A303, S304, A305, F306, M307, G308, M309, S310,and Q311.

The corresponding epitopes and N protein sequences as described by Shiet al., supra are also incorporated by reference.

IgY antibodies that bind to one of the above combinations of N proteinresidues may be characterized as antibodies that recognizeconformational epitopes; see Jiandong Shi, et al., supra.

The invention also pertains to a diagnostic method using a mixture oftwo, three, four or more monospecific antibodies recognizing differentepitopes or domains of the N protein.

Another aspect of this technology is directed to an antibody conjugatecomprising any of the antibodies disclosed herein including to amonospecific polyclonal IgY antibody that binds to the N terminal domainof MERS-CoV that is conjugated to at least one detectable marker or to atherapeutic agent; or to an antibody conjugate comprising a monospecificpolyclonal IgY antibody that binds to the LKR of MERS-CoV that isconjugated to at least one detectable marker or to a therapeutic agent;and/or to an antibody conjugate comprising a monospecific polyclonal IgYantibody that binds to the C terminal domain of MERS-CoV that areconjugated to at least one detectable marker or to a therapeutic agent.The IgY antibodies, or their antigen binding fragments and conjugatesmay also be attached to a solid substrate such as a microtiter platewell or to chromatography beads or other substrates such as woven ornon-woven materials or to a plastic, metal, silicon, or glass surface.

Another aspect of this technology is a method for treating a subject atrisk of being infected by, or infected by, MERS-CoV comprisingadministering an IgY antibody, chimeric IgY antibody or humanized IgYantibody to the subject. In one embodiment, the method comprisesadministering an IgY composition or pharmaceutical compositionintravenously, intramuscularly, topically, intradermally,intramucosally, subcutaneously, sublingually, orally, intravaginally,intracavernously, intraocularly, intranasally, intrarectally,gastrointestinally, intraductally, intrathecally, subdurally,extradurally, intraventricular, intrapulmonary, into an abscess, intraarticularly, into a bursa, subpericardially, into an axilla,intrauterine, into the pleural space, intraperitoneally, transmucosally,or transdermally. Advantageously, a humanized IgY composition typicallycomprising one or more humanized monoclonal antibodies, such as an IgGantibody having its CDRs replaced with IgY CDRs, or an IgY antibodyhaving its non-CDR regions replaced by IgG regions, may be administeredinto the nasal cavity, sinuses, or upper or lower respiratory system.

The foregoing paragraphs have been provided by way of generalintroduction, and are not intended to limit the scope of the followingclaims. The described embodiments, together with further advantages,will be best understood by reference to the following detaileddescription taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Comparison of IgY and IgG structures.

FIG. 2. Components of MERS-CoV.

FIG. 3. Illustration of some steps of production and purification of IgYantibodies in avians and use of IgY antibodies in antibody-based assayfor MERS-CoV N protein.

FIG. 4. Illustration of one embodiment of an immunoswab assay for Nprotein.

FIG. 5. Affinity purification of monospecific antibodies.

DETAILED DESCRIPTION OF THE INVENTION

The enzyme-linked immunosorbent assay (ELISA) is a commonly usedanalytical biochemistry assay. The assay uses a solid-phase type ofenzyme immunoassay (EIA) to detect the presence of a ligand in a liquidsample using antibodies directed against the ligand to be measured. Inthe present invention, an IgY-based ELISA is used to detect a proteinligand, MERS-CoV N protein.

In one simple form of an ELISA, a sample to be tested for a particularantigen such as MERS-CoV N protein is attached to a surface such as tothe bottom of a microtiter plate well. An antigen-specific IgY antibody,which has been linked to an enzyme, is applied over the surface so itcan bind the antigen. Unbound IgY antibodies are removed. In the finalstep, a substance containing the enzyme's substrate is added. If thereis binding between the MERS-CoV N protein and the IgY antibody thesubsequent reaction produces a detectable signal, most commonly a colorchange.

Performing an ELISA involves at least one IgY antibody with specificityfor MERS-CoV N protein antigen. The sample with an unknown amount ofantigen is immobilized on a solid support (usually a polystyrenemicrotiter plate) either non-specifically (via adsorption to thesurface) or specifically (via capture by another antibody specific tothe same antigen, in a “sandwich” ELISA). After the antigen isimmobilized, an antigen-specific detection antibody is added, forming acomplex with the antigen. The detection antibody can be covalentlylinked to an enzyme or can itself be detected by a secondary antibodythat is linked to an enzyme through bioconjugation. Between each step,the plate is typically washed with a mild detergent solution to removeany proteins or antibodies that are non-specifically bound. After thefinal wash step, the plate is developed by adding an enzymatic substrateto produce a visible signal, which indicates the quantity of antigen inthe sample.

Diagnostic samples for ELISA may be obtained from subjects exposed to orinfected with MERS-CoV. Subjects include humans such as a male or femalechild less than reproductive age, a male or female of reproductive age,a pregnant female, or an adult. The subject may be one at risk ofexposure to MERS-CoV such as one who works in proximity to infectedhumans or animals or who is exposed to animal products like milk ormeat, animal fluids such as saliva or blood, or animal waste productssuch as urine or feces, or a human or animal otherwise at risk of, orthat is, infected with MERS-CoV.

Samples may also be acquired from subjects with impaired respiratorysystems including pneumonia, cystic fibrosis, tuberculosis, asthma,emphysema, bronchitis, smokers, lung cancer, pneumoconiosis, and chronicbronchitis and other forms of COPD, autoimmune diseases, or from thoseat risk of immune dysfunction or immunodeficiency. In some preferredembodiments, a sample is collected by nasopharyngeal swab,nasopharyngeal aspirate, nasal wash, or nasopharyngeal wash.

Subjects for diagnosis. A subject from whom a biological sample is takenfor diagnostic testing may be a human or be a non-human animal such as amember of the Camelidae family, such as a dromedary or a Bactrian camelor a llama, alpaca, vicuña, or guanaco. A subject may also be a pet(e.g., dog or cat), livestock (e.g., horse, cattle, goats, sheep, pigs)or wild animal (e.g., bats, mice, rats) susceptible to infection withMERS-CoV or which act as a vectors, carriers or reservoirs for thevirus. The term subject includes both adult and juvenile and male andfemale subjects.

In addition to the human an animal subjects mentioned above from whomsamples may be taken, subjects at risk of an active vaccine-induced lungpathology may be selected for passive immunization to avoid theside-effects of administering an immunogenic viral protein.

The method as disclosed herein may be used with antibody (IgY) basedassays. Some preferred types of assays include ELISA (indirect or directcompetitive ELISA sandwich ELISA, direct or indirect ELISA),immunodiffusion, immunochromatography assay, western blotting, andimmunohistochemistry assay including immunofluorescence.

Additional types or details of ELISA assays for use with IgY asdisclosed herein are described below.

Direct ELISA. The steps of direct ELISA substantially follow themechanism below: A buffered solution of the antigen to be tested for isadded to each well (usually 96-well plates) of a microtiter plate, whereit is given time to adhere to the plastic through charge interactions. Asolution of nonreacting protein, such as bovine serum albumin or casein,is added to each well in order to cover any plastic surface in the wellwhich remains uncoated by the antigen. The primary antibody with anattached (conjugated) enzyme is added, which binds specifically to thetest antigen coating the well. A substrate for this enzyme is thenadded. Often, this substrate changes color upon reaction with theenzyme. The higher the concentration of the primary antibody present inthe serum, the stronger the color change. Often, a spectrometer is usedto give quantitative values for color strength.

The enzyme acts as an amplifier; even if only few enzyme-linkedantibodies remain bound, the enzyme molecules will produce many signalmolecules. Within common-sense limitations, the enzyme can go onproducing color indefinitely, but the more antibody is bound, the fasterthe color will develop. A major disadvantage of the direct ELISA is thatthe method of antigen immobilization is not specific; when serum is usedas the source of test antigen, all proteins in the sample may stick tothe microtiter plate well, so small concentrations of analyte in serummust compete with other serum proteins when binding to the well surface.The sandwich or indirect ELISA provides a solution to this problem, byusing a “capture” antibody specific for the test antigen to pull it outof the serum's molecular mixture

ELISA may be run in a qualitative or quantitative format. Qualitativeresults provide a simple positive or negative result (yes or no) for asample. The cutoff between positive and negative is determined by theanalyst and may be statistical. Two or three times the standarddeviation (error inherent in a test) is often used to distinguishpositive from negative samples. In quantitative ELISA, the opticaldensity (OD) of the sample is compared to a standard curve, which istypically a serial dilution of a known-concentration solution of thetarget molecule. For example, if a test sample returns an OD of 1.0, thepoint on the standard curve that gave OD=1.0 must be of the same analyteconcentration as the sample.

The use and meaning of the names “indirect ELISA” and “direct ELISA”differs in the literature and on web sites depending on the context ofthe experiment. When the presence of an antigen is analyzed, the name“direct ELISA” refers to an ELISA in which only a labelled primaryantibody is used, and the term “indirect ELISA” refers to an ELISA inwhich the antigen is bound by the primary antibody which then isdetected by a labeled secondary antibody. In the latter case a sandwichELISA is clearly distinct from an indirect ELISA. When the “primary”antibody is of interest, e.g. in the case of immunization analyses, thisantibody is directly detected by the secondary antibody and the term“indirect ELISA” applies to a setting with two antibodies.

Sandwich ELISA. A plate is coated with a capture antibody; a sample isadded, and any antigen present binds to capture antibody; a detectingantibody is added, and binds to antigen; an enzyme-linked secondaryantibody is added, and binds to detecting antibody then substrate isadded, and is converted by enzyme to detectable form. A “sandwich” ELISAis used to detect sample antigen. The steps are: A surface is preparedto which a known quantity of capture antibody is bound. Any nonspecificbinding sites on the surface are blocked. The antigen-containing sampleis applied to the plate, and captured by antibody. The plate is washedto remove unbound antigen. A specific antibody is added, and binds toantigen (hence the ‘sandwich’: the antigen is stuck between twoantibodies). This primary antibody could also be in the serum of a donorto be tested for reactivity towards the antigen. Enzyme-linked secondaryantibodies are applied as detection antibodies that also bindspecifically to the antibody's Fc region (nonspecific). The plate iswashed to remove the unbound antibody-enzyme conjugates. A chemical isadded to be converted by the enzyme into a color or fluorescent orelectrochemical signal. The absorbance or fluorescence orelectrochemical signal (e.g., current) of the plate wells is measured todetermine the presence and quantity of antigen. Without the first layerof “capture” antibody, any proteins in the sample (including serumproteins) may competitively adsorb to the plate surface, lowering thequantity of antigen immobilized. Use of the purified specific antibodyto attach the antigen to the plastic eliminates a need to purify theantigen from complicated mixtures before the measurement, simplifyingthe assay, and increasing the specificity and the sensitivity of theassay. A sandwich ELISA used for research often needs validation becauseof the risk of false positive results.

Competitive ELISA. Another use of ELISA is through competitive binding.The steps for this ELISA are somewhat different from the first twoexamples: unlabeled antibody is incubated in the presence of its antigen(sample). These bound antibody/antigen complexes are then added to anantigen-coated well. The plate is washed, so unbound antibodies areremoved. (The more antigen in the sample, the more Ag-Ab complexes areformed and so there are less unbound antibodies available to bind to theantigen in the well, hence “competition”.) The secondary antibody,specific to the primary antibody, is added. This second antibody iscoupled to the enzyme. A substrate is added, and remaining enzymeselicit a chromogenic or fluorescent signal. The reaction is stopped toprevent eventual saturation of the signal. Some competitive ELISA kitsinclude enzyme-linked antigen rather than enzyme-linked antibody. Thelabeled antigen competes for primary antibody binding sites with thesample antigen (unlabeled). The less antigen in the sample, the morelabeled antigen is retained in the well and the stronger the signal.Commonly, the antigen is not first positioned in the well.

Immunoswab. One embodiment of this assay is incorporated by reference toKammila, S, et al. A rapid point of care immunoswab assay for SARS-CoVdetection.” JOURNAL OF VIROLOGICAL METHODS, 2008, 152(1-2): 77-84. Thisassay may be performed with the IgY antibodies disclosed herein. FIG. 3describes several variants of this method. Briefly, a capture antibodythat recognizes MERS-CoV N protein is bound to a swab, such as a calciumalginate swab, unbound antibody is rinsed off, unbound sites arequenched with 2% BSA or other protein. For detection of N protein, abiological sample suspected to containing N protein is applied to theswab. Unbound material is washed off and an enzyme-linked detectingantibody which binds to N protein is applied. Unbound antibody is washedoff and binding or the amount of binding is detected by action of anenzyme bound to the detecting antibody on a substate. Preferably, boththe capture and detecting antibodies are IgY, which may be monoclonal,monospecific, or polyspecific IgY. In some embodiments, the detectingantibody comprises biotin and the enzyme comprises strepavidin andhorseradish peroxidase and the substrate for the enzyme is3,3′,5,5′-Tetramethylbenzidine or TMB. A similar system has been used todetect SARS N protein. In view of the structural and immunologicaldifferences among various Coronavirus N proteins, one objective was todetermine whether this system would provide high specificity andsensitivity for detection of MERS-CoV N protein and which IgY antibodiesshould be selected for such an assay.

One skilled in the art can select an appropriate enzymatic marker forELISA. Enzymatic markers for ELISA include OPD (o-phenylenediaminedihydrochloride) turns amber to detect HRP (Horseradish Peroxidase),which is often used to as a conjugated protein. TMB(3,3′,5,5′-tetramethylbenzidine) turns blue when detecting HRP and turnsyellow after the addition of sulfuric or phosphoric acid. ABTS(2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt)turns green when detecting HRP. PNPP (p-Nitrophenyl Phosphate, DisodiumSalt) turns yellow when detecting alkaline phosphatase.

MERS-CoV neutralization assay may be performed using the IgY antibodyproducts disclosed herein. Such assays are known in the art and can beadapted to use of IgY antibodies that recognize MERS-CoV N protein orits epitopes. Such assays are also incorporated by reference to Amanat,F., et al., An in vitro microneutralization assay for SARS-CoV-2serology and drug screening., CURRENT PROTOCOLS, 2020, 58(1). Suchassays may be used to detect levels of neutralizing antibodies in asample, identify neutralizing epitopes on N protein, or identify CDRsuseful for making chimeric or humanized antibodies.

Other tests and assays for detection of MERS-CoV or MERS-CoV N proteinwith IgY. These include immunochromatographic, western blotting,immunoenzymatic, immunofluorescence tests or tests performed usingimmunosensors.

In addition to the immunodiagnostic methods disclosed herein, the IgYantibody products disclosed herein may be used as immunotherapeutics.

Nucleocapsid or N protein. The N protein of MERS-CoV is a phosphorylatedbasic protein and is the second largest structural protein, containing413 amino acid residues. The N protein binds to the RNA genome to form anucleocapsid, which is important for virus replication. The NP is themost abundantly expressed antigen among the other MERS-CoV antigens andamino-acid mutations are uncommon. SEQ ID NO: 1 describes an MERS-CoV Nprotein sequence and SEQ ID NOS: 2 and 3 segments of the N protein.MERS-CoV N protein functions as an IFN antagonist. It suppressesRIG-I-induced type I and type III IFN production by interfering withTRIM25-mediated RIG-I ubiquitination. Antibodies against the N proteinmay appear before antibodies against the S protein. Thus, the inventorsbelieve that sensitive detection of N protein in bodily fluids isimportant for diagnosis or for assessing host response to viralinfection. Other structural and functional features of MERS-CoV Nprotein are incorporated by reference to Li, Y. et al., MolecularCharacteristics, Functions, and Related Pathogenicity of MERS-CoVProteins, ENGINEERING, 2019, 5(5), 940-947.

IgY. In chickens, immunoglobulin Y is often compared to Immunoglobulin G(IgG). Like IgG, it is composed of two light and two heavy, but has ashorter heavy chain the IgG. The steric flexibility of the IgY moleculeis less than that of IgG.

IgY passes from an immunized hen's blood to her eggs and extraction ofthe IgY from egg yolks provides simple and non-invasive alternative forantibody production. Production of IgY in high yields compared toproduction of IgG antibodies in mammals, such as rabbits provides anadvantage. One chicken can produce about 22 gr of IgY antibodies with2-10% of the antibodies being target (immunogen) specific. This exceedsthe production of IgG from four rabbits. IgY antibodies can be stored ineggs at 4° C. for at least one year.

IgYs generally have no interactions with mammalian immune components orassay materials used to detect mammalian antibodies. These includenon-reactivity with cellular Fc receptors, mammalian IgG, rheumatoidfactor, mammalian complement components, protein A, and protein G. Thislack of reactivity helps reduce the occurrence of false positive resultsin assays employing IgY instead of IgG.

In addition, the avian IgY repertoire differs from mammalian repertoirepermitting IgY to bind to epitopes not recognized, or poorly recognizedby mammalian antibodies.

IgY antibodies may have K_(D) or average K_(D) values ranging frommicomolar (10⁻⁶ L/mol), to nanomolar (10⁻⁷-10⁻⁹ L/mol). High affinityIgY antibodies may have K_(D)s in the picomolar range up to 10⁻¹² L/mol.Binding affinity is typically measured and reported by the equilibriumdissociation constant (K_(D)), which is used to evaluate and rank orderstrengths of bimolecular interactions. In biochemistry or pharmacology,protein-ligand complex having high affinity if the Kd is below 100 nM(for antibody-antigen complex below 10 nM), medium affinity in the range100 nM-10 uM, and low affinity if the Kd is above 10 uM. IgY antibodiesas disclosed herein may have K_(D) values in the low micromolar (about10⁻⁶) to nanomolar (about 10⁻⁷, 10⁻⁸ to 10⁻⁹) range or in the lownanomolar range of about 10⁻⁹, 10⁻¹⁰ or 10⁻¹¹ with very high affinityantibodies being in the picomolar (10⁻¹²) range.

In some embodiments chicken IgY exhibits high avidity of about 10⁻⁹L/mol. Avidity is the strength of interaction between a multivalentantibody and epitopes.

The inventors considered that chicken IgY polyclonal antibodies wouldhave higher avidity, higher specificity and lower cross-reactivity ascompared to IgG antibodies, that they could have a broaderantigen-binding host range, due to the great evolutionary distancebetween chickens and mammals, and that they could exhibit high avidity(10⁻⁹ L/mol) even after the first immunization. Moreover, the inventorsconsidered that the response of immunoglobulins in chickens to thehighly conserved mammalian proteins would be robust showing higheraffinity, thereby potentially targeting broad spectrum of epitopes onprotein immunogens.

In some embodiments, monospecific IgY antibodies are eluted from anaffinity column and purified away from other antibodies and components.Affinity chromatography is a method used for purification of a specificmolecule or group of molecules from a mixture. By using the relationshipbetween two molecules, such as the affinity between an antigen andantibody, one can purify the desired biological molecules.

Most methods disclosed herein may employ naturally occurring IgYantibodies such as those produced by exposure or immunization of achicken or other avian to MERS-CoV N protein or its epitopes. In someembodiments, these are isolated from the egg yolks of immunized avians.These are typically polyclonal antibodies, however antigenic specificityof the polyclonal antibodies may be tailored by choice of immunizationwith the entire N protein, denatured or soluble N protein, N proteinquaternary forms (e.g., dimers or tetramers), or by immunization with Nprotein fragments or peptide epitopes. Alternatively, monoclonalMERS-CoV IgY antibodies may be used. These too may be directed todifferent linear or conformational epitopes of N protein.

Monospecific antibodies are antibodies whose specificity to antigens issingular in any of several ways: antibodies that all have affinity forthe same antigen; antibodies that are specific to one antigen or oneepitope; or antibodies specific to one type of cell or tissue.Monoclonal antibodies are monospecific, but monospecific antibodies mayalso be produced by other means than producing them from a common germcell, for example, by immunization with a specific peptide epitope orother segment of a longer protein or by affinity purification to asubstrate comprising an epitope of interest.

Immunization. Methods for immunizing an animal such as a chicken orother avian are known in the art and are incorporated by reference toLABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY, 1966, 19,131-144, see Chapter 4, Immunization with peptides. Preferably, theavians are inbred or genetically identical. In some methods adjuvantsare used to boost production of IgY antibodies. An adjuvant is apharmacological or agent that modifies the effect of other agents.Adjuvants may be added to a protein construct as disclosed herein, suchas a conjugate comprising N protein epitopes and a carrier protein, toboost the humoral or cellular immune responses and produce more anti-IgYantibodies and longer-lasting immunity, thus minimizing the dose ofprotein construct needed. Adjuvants that may be compounded with, orotherwise used along with N protein, its fragments or peptide epitopesdisclosed herein include, but are not limited to, inorganic compoundsincluding alum, aluminum hydroxide, aluminum phosphate, calciumphosphate hydroxide; mineral oil or paraffin oil; bacterial products ortheir immunologically active fractions, such as those derived killedBordatella pertussis, Mycobacterium bovis, or bacterial toxoids;organics such as squalene; detergents such as Quil A, saponins such asQuillaja, soybean or polygala senega; cytokines such as IL-1, IL-2 orIL-12; Freund's complete adjuvant or Freund's incomplete adjuvant; andfood based oils like Adjuvant 65, which is a product based on peanutoil. Those skilled in the medical or immunological arts may select anappropriate adjuvant based on the type of patient and mode ofadministration of the protein construct of the invention.

Affinity purification. IgY antibodies may be affinity purified to Nprotein, its fragments, or epitopes using methods known in the art. Suchmethods are also incorporated by reference to Hnasko, et al., AffinityPurification of Antibodies, METHODS MOL BIOL , 2015, 1318, 29-41, toDarcy, et al., Purification of Antibodies Using Affinity Chromatography,METHODS MOL BIOL., 2017;1485, 305-318; or to Arora, et al., Affinitychromatography for antibody purification, METHODS MOL BIOL.2014;1129:497-516. One example of affinity purification of antibodies isshown in FIG. 5.

Regarding antibodies, monospecific and monovalent overlap in meaning;both can indicate specificity to one antigen, one epitope, or one celltype (including one microorganism species). However, antibodies that aremonospecific to a certain tissue, or all monospecific to the same tissuebecause clones, can be polyvalent in their epitope binding.

Monoclonal IgY antibodies to MERS-CoV N protein may be produced bymethods known in the art or may be custom ordered to an antigen ofinterest commercially, for example, from Creative Biolabs, worldwideweb.creative-biolabs.com/igy-antibody-generation.html (last accessedOct. 2, 2020. One typical method for production of IgY monoclonalantibodies comprises PCR amplification of chicken genes encoding the IgYantibody repertoire followed by construction of a phage library. Thephage library is then screened for positive antibody binders for Nprotein epitopes. Antibodies that bind to N protein are then expressedin E. coli, isolated and optionally further characterized as toaffinity, avidity, stability and specificity. Monoclonal fragments, suchas monoclonal scFv or Fab types of IgY antibody, may be produced in asimilar manner after phage display library screening. Chimeric orhumanized IgY may be produced by transferring the scFv regions of theIgY monoclonal antibodies into a human IgG expression vector andmanufacturing the chimeric antibody in CHO cells.

Chicken antibody repertoires focused on a number of defined targets,such as MERS-CoV N protein or its epitopes, can be constructed usinglymphocyte mRNA from chickens immunized with a single epitope or mixtureof several different N protein epitopes. Immune responses to each of theindividual epitopes can be determined by extracting egg-yolk IgY andtesting for antigen-specific antibodies using ELISA. The chickensplenocytes are then recovered, RNA is extracted, and after reversetranscription, the immunoglobulin V_(H) and V_(L) regions are amplifiedby PCR and joined via a single glycyl residue for surface expression ona collection of filamentous bacteriophages. The resulting displaylibrary is screened by panning to isolate binders.

A “carrier” or “excipient”, as used herein, includes any and allsolvents, dispersion media, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, solid binders,lubricants and the like useful for the diagnostic assays disclosedherein. This term also includes pharmaceutical carriers and excipientssuch as those described by and incorporated by reference to Remington'sThe Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro,(Lippincott, Williams & Wilkins, Baltimore, Md., 2006). Any suitablebuffer, carrier or excipient may be combined with the N protein-bindingIgY, antigen-binding IgY portions. IgY CDRs, or IgY conjugates whichbind to N protein; or with biological samples suspected of containing Nprotein.

Kits. In some embodiments, kits in accordance with the presentdisclosure may be used to detect, diagnose or for prognosis of MERS-CoVrelated disease, disorder or condition in a subject. In someembodiments, the kits comprise a container comprising one or a pluralityof pharmaceutical compositions comprising IgY specific for N protein,compositions described herein and, optionally, a device used toadminister the one or more pharmaceutical compositions. The disclosureprovides a kit to perform any of the methods or assays described herein.In some embodiments, the kit comprises at least one container comprisinga therapeutically effective amount of one or a plurality of N proteinspecific IgYs. In some embodiments, the IgY or IgY-based products suchas conjugates are present in solution such as a buffer with adequate pHand/or other necessary additive to minimize degradation or aggregationof IgY during prolonged storage. In some embodiments, the IgYs arelyophilized for the purposes of resuspension after prolonged storage. Insome embodiments, the kit optionally comprises instructions to performany or all steps of any method described herein.

The kit may contain two or more containers, packs, or dispenserstogether with instructions for preparation of an array. In someembodiments, the kit comprises at least one container comprising theIgYs described herein and a second container comprising a means formaintenance, use, and/or storage of the IgY such as storage buffer orsecondary antibodies that bind to IgY or that bind to N protein orindicators for antibody binding. In some embodiments, the kit comprisesa composition comprising any IgY disclosed herein in solution orlyophilized or dried and accompanied by a rehydration mixture. In someembodiments, the IgYs and rehydration mixture may be in one or moreadditional containers.

The compositions included in the kit may be supplied in containers ofany sort such that the shelf-life of the different components arepreserved, and are not adsorbed or altered by the materials of thecontainer. For example, suitable containers include simple bottles thatmay be fabricated from glass, organic polymers, such as polycarbonate,polystyrene, polypropylene, polyethylene, ceramic, metal or any othermaterial typically employed to hold reagents or food; envelopes, thatmay consist of foil-lined interiors, such as aluminum or an alloy. Othercontainers include test tubes, vials, flasks, and syringes. Thecontainers may have two compartments that are separated by a readilyremovable membrane that upon removal permits the components of thecompositions to mix. Removable membranes may be glass, plastic, rubber,or other inert material.

Kits may also be supplied with instructional materials. Instructions maybe printed on paper or other substrates, and/or may be supplied as anelectronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, zipdisc, videotape, audio tape, or other readable memory storage device.Detailed instructions need not be physically associated with the kit;instead, a user may be directed to an internet web site specified by themanufacturer or distributor of the kit, or supplied as electronic mail.

In one embodiment, a kit comprises 96-well ELISA plates precoated withmonospecific anti-NP IgY antibodies, washing buffer (PBS Tween), skimmedmilk in PBST as blocking buffer, monoclonal antibodies specific for NP,HRP (horse radish peroxidase)-conjugate for detection of boundedantibodies, and a substrate O-phenylendiamine dihydrochloride fordetection of the reaction.

EXAMPLE 1 Immunization and Collection of Eggs Containing IgY

Twenty Lohmann laying hens which are 25 weeks old are bought from alocal broiler farm. The animals are placed in cages, which are dedicatedto broiler chickens, in groups of two animals per cage under a regimenof a 12 hr:12 hr light-dark cycle at an ambient temperature of 24±3° C.and at a relative humidity of 75±5%. Water and commercial food areoffered ad libitum. Further description of Lohmann laying hens and othersuitable chickens is available at hypertext transferprotocol://frabopoultry.com/wp-content/uploads/2016/10/Lohmann_LSL-Lite.pdf(last accessed Jan. 21, 2021, incorporated by reference).

Hens are divided into two immunization Groups A and B each with tenhens.

As detailed below, the ten hens in an immunization Group A are injectedwith 200 μg of a commercially available recombinant MERS-CoV N proteinin PBS (phosphate buffered saline) in combination with an adjuvant andthe ten hens in a control Group B are injected only with PBS and theadjuvant.

For Group A, recombinant N protein is emulsified at a ratio of 1:1 incomplete Freund's Adjuvant (“CFA”; Sigma) and for boosts in IncompleteFreund's Adjuvant (“IFA”; Sigma) by pipetting the mixture up and downthrough a 19 gauge needle attached to a 5 ml syringe until a stableemulsion is formed.

On day 0 hens in Group A are immunized on the left and right sidepectoral muscles with 200 μg of recombinant N protein emulsified withcomplete Freund's Adjuvant (“CFA”; Sigma).

On days 12 and 28, the Group A hens are boosted on the left and rightside pectoral muscles with 200 μg of recombinant N protein emulsified ata ratio of 1:1 with incomplete Freund's Adjuvant (“IFA”; Sigma).

Group B hens are injected with PBS emulsified with CFA (day 0) or IFA(days 12 and 28) but no N protein on the same days as hens in Group A.

To determine antibody responses, blood samples are taken from hens inboth Groups A and B before each immunization and on the last day beforeslaughter of the hens.

Eggs from hens in both groups are collected daily one week before and 24hours after initial immunization. Egg collection is continued for 12weeks. Eggs are stored at 4° C. prior to isolation of IgY isolation fromthe egg yolks.

IgY Separation and Purification

IgY antibodies are purified from stored eggs of Groups A and B using anEGGstract® IgY purification system from PROMEGA® according tomanufacturer's instructions. Briefly, the yolks of the eggs areseparated from the egg whites using an egg separator. Then a 14%polyethylene glycol (PEG6000) solution is added to the yolks atvolumetric ratio 3:1. After 30 min stirring at ambient temperature (RT),the mixture is centrifuged at 5,000 g at 10° C. for 20 min. Thesupernatant is collected and filtered through sterile gauze.

PEG6000 is added to the filtered supernatant with gentle stirring.

The mixture is then centrifuged and 5,000 g for 20 mins.

The supernatant is collected and solid ammonium sulfate is added toprecipitate proteins in the mixture which is stirred at 40° C. for 24hrs.

The precipitate is collected by centrifugation and washed with asolution of saturated ammonium sulfate.

The washed precipitate is then dialyzed against PBS and freeze dried toobtain a powder which is stored at −20° C.

To assess the purity and molecular weight of IgY in the freeze-driedpowder, samples of the power are suspended in a sodium dodecylsulfate-polyacrylamide gel electrophoresis (“SDS-PAGE”) buffer andmolecular components are resolved by SDS-PAGE under reducing conditionsusing a 12% polyacrylamide gel with a Mini-PROTEAN® 3 cell (BIO-RAD®Laboratories, USA). The samples are mixed with 2X sample buffer boiledfor 10 min at 100° C. 25 μl of the purified IgY is loaded into eachwell. Prestained Blue Protein Markers are used as molecular weightmarkers. Electrophoresis is performed at room temperature in runningbuffer (Tris-Glycine buffer) at 200 volts for 40 min. The protein bandsare then visualized with Coomassie Brilliant Blue stain and analyzedusing special software.

EXAMPLE 3 Determination of Reactivity of Anti-NP IgY Antibodies by ELISA

The reactivity of samples of anti-N-protein IgY antibodies obtained asdescribed above is determined by ELISA. Briefly, recombinant MERS-CoV Nprotein at a concentration of 2.5 mg/mL is contacted with ELISA platesovernight to coat the plates with the N protein. Unbound sites areblocked with skim milk after which the plates are washed. After washing100 uL/well of the anti MERS-CoV yolk antibodies of the NP-immunizedGroup A and yolk antibodies from adjuvant control Group B are incubatedin the plates for 90 mins and then washed.

After washing a conjugate antibody that binds to IgY antibodies is addedfor detection of IgY bound to the N protein on the plates.

A colorimetric substrate is used to visually determine an amount ofantibody binding to the plates.

Color intensity is measured by ELISA reader and correlates with amountof N protein IgY antibodies in samples.

EXAMPLE 4 Western Blotting Assay

Western blotting is performed to check the specificity of the antiMERS-COV NP IgY antibodies.

MERS-CoV N protein is fractionated on 8-12% (SDS-PAGE) and transferredto polyvinylidene difluoride (PVDF) membrane (Milipore Ltd.).

The membrane is then washed for 10 minutes in 1X tris buffered salinewith 0.1% Tween-20 (TBS-T) and blocked overnight with 5% skim milk inTBS-T. Membranes are washed for 1 hour at 10-minute intervals in TBS-T.

Subsequently, the membrane is incubated with anti MERS-CoV yolkantibodies of the first and second groups and yolk antibodies fromadjuvant control group for 1 hour at RT is followed by incubation withHRP-labeled goat anti-chicken IgG.

Protein detection is carried out using an HRP colorimetric detectionsystem.

EXAMPLE 5 Detection of MERS-CoV Infected Cells by FluorescentImmunocytochemistry

MERS-CoV is inoculated on Vero cells and infected cells are harvestedafter 48 hours. The cells are collected by centrifugation at 1500 for 5minutes and washed twice with wash buffer.

100 μl of the cells are added to tubes containing 200 μ1 l of blockingbuffer and are incubated for 1 hour at room temperature and then washedtwice with wash buffer.

200 μl of the diluted IgY antibodies are added and incubated for 1 hourat room temperature and then washed twice with wash buffer.

100 μl of FTIC conjugated anti-chicken antibodies are added in a 1:2500dilution and incubated for 1 hour at room temperature and then washedtwice with wash buffer.

30 μl of the cell suspension is fixed on a slide and is observed underfluorescent microscope. Fluorescence indicates the presence MERS-CoV.

EXAMPLE 6 Detection of MERS-COV in a Clinical Sample by Sandwich ELISA

Nasopharyngeal swabs from camels that have been confirmed infected byMERS-CoV by RT-PCR are obtained using commercially available UniversalTransport Medium.

Samples are tested for the presence of, or the concentration of,MERS-CoV using ELISA in 96 well microtiter plates (Nunc).

Microtiter plates are coated with anti-NP IgY antibodies as capturingantibody in a bicarbonate buffer (pH 9.6).

Plates are then incubated overnight at 4° C. and blocked with 5% non-fatskim milk followed by washing with PBST.

Plates are incubated with clinical samples overnight at 4° C. (50 μl perwell). Clinical samples previously treated with 100 μl of RIPA(radioimmunoprecipitation assay) buffer for protein extraction areincubated at 37° C. for an hour.

Rabbit anti-NP antibodies (previously prepared by the inventors) areadded in an appropriate dilution as revealing antibody.Peroxidase-conjugated anti-rabbit secondary antibodies raised in goatare used as secondary antibodies.

Plates are then thoroughly washed and developed with a chromogenicsubstrate with the reaction stopped by addition of H₂SO₄. Absorbance ismeasured at 450 nm using microtiter plate reader where absorbancecorresponds to presence or concentration of MERS-CoV in a sample.

EXAMPLE 7 PCR amplification of Gene Products of Heavy and Light IgYChains

Seven days following a final immunization with N protein, chickens areeuthanized, and spleens are harvested. Spleens are placed immediately inTrizol reagent for homogenization. The extraction of total RNAs iscarried out and reversely transcribed to synthesize the first-strandcDNA using a Super Script RT kit (Invitrogen). After PCR amplificationwith chicken specific primers, gene products of heavy and light chainvariable regions (VH and VL) are obtained and may be sequenced. The VHand VL sequences may be used to engineer antibodies by methods known inthe art (for example, by a commercial service) which recognize MERS-CoVepitopes. Once the heavy- and light-chain sequences are determined,antibodies that recognize MERS-CoV can be specific modified orengineered to suit a particular diagnostic or therapeutic application,for example, to increase sensitivity or specificity or to adapt them totherapeutic administration. In some embodiments, an IgY antibody or VLor VH sequence may be humanized or adapted to administration to acamelid.

Definitions.

The terms “prevention”, “prevent”, “preventing”, “prophylaxis” and asused herein refer to a course as disclosed herein, initiated prior tothe onset of a clinical manifestation of a disease state or condition,including detection of MERS-CoV, its antigens or nucleic acids, orcellular or humoral responses to MERS-CoV so as to prevent or reducesuch clinical manifestation of the disease state or condition.Prevention of reduction in severity of a clinical manifestation need notbe absolute to be useful, for example, it may constitute a reduction inthe manifestation of >0, 5. 10, 20, 30, 40, 50. 60, 70, 80. 90, <100 or100%

The terms “treatment”, “treat” and “treating” as used herein refers acourse of action such as administering an IgY-based compound orIgY-based pharmaceutical composition initiated after the onset of aclinical manifestation of a disease state or condition so as toeliminate or reduce such clinical manifestation of the disease state orcondition. Such treating need not be absolute to be useful.

The term “in need of treatment” as used herein refers to a judgment madeby a caregiver that a patient requires or will benefit from treatment.This judgment is made based on a variety of factors that are in therealm of a caregiver's expertise, but that includes the knowledge thatthe patient is ill, or will be ill, as the result of a condition that istreatable by a method, IgY antibody product or pharmaceuticalcomposition of the disclosure.

The term “in need of prevention” as used herein refers to a judgmentmade by a caregiver that a patient requires or will benefit fromprevention. This judgment is made based on a variety of factors that arein the realm of a caregiver's expertise, but that includes the knowledgethat the patient will be ill or may become ill, as the result of acondition that is preventable by a method, an IgY-based product, orpharmaceutical composition of the disclosure.

The term “individual”, “subject” or “patient” as used herein refers to ahuman, camel including dromedary camels, bat, or other animalsusceptible to infection by MERS-CoV or as a vector or carrier ofMERS-CoV. It can also refer to chickens, turkeys, ducks, geese,ostridges, emus, or other avians capable of production of IgY. The termmay specify male or female or both, or exclude male or female. In oneaspect, the patient is an adult human. In another aspect, the patient isa non-neonate human infant. In another aspect, the patient is a humantoddler, child, adolescent, or adult at least 16, 17, 18, 21, 25, 30,40, 50, 60, 70, 80 90 or 100 years old.

The term “neonate”, or newborn, refers to an infant in the first 28 daysafter birth. The term “non-neonate” refers to an animal older than 28days. The methods disclosed herein are applicable to neonates ornon-neonates.

The term “effective amount” as used herein refers to an amount of anagent, either alone or as a part of a pharmaceutical composition, thatis capable of having any detectable, positive effect on any symptom,aspect, or characteristics of a disease state or condition. Such effectneed not be absolute to be beneficial.

The term “immunize”, “actively immunize”, “actively immunizing”, and“active immunization” means to purposefully immunize a subject, such asan avian, by exposing a subject to an antigen, for example, an antigenderived from MERS-CoV; such exposure may be carried out by exposing thesubject to an intact organism, an attenuated organism, a portion of theorganism such as N protein or an immunogenic segment of N protein, or acombination of the foregoing.

The term “passively immunize”, “passively immunizing”, and “passiveimmunization” means to provide antibodies against an antigen, forexample, an antigen derived from MERS-CoV N protein to a subject withoutnecessarily eliciting an immune response to the organism in the subject.Passive immunization provides immediate protection but the subject neednot develop memory cells as a result.

The term “passive immunity” as used herein refers to artificiallyacquired immunity achieved by the transfer of antibodies to the subject.

The terms “egg” or “egg product” each mean an avian sourced whole shellegg, unfertilized egg, fertilized egg, unhatched embryo, or any productor fraction derived therefrom including egg white or egg yolk. Aviansinclude chickens, turkeys, ducks, geese, emus, and ostriches.

The term “antigen” refers to an entity or fragment thereof which caninduce an immune response in an organism, particularly an animal. Theterm includes immunogens and regions thereof responsible forantigenicity or antigenic determinants. Antigenic determinants includepeptides of sufficient length to present via MHC-I or MHC II moleculesor peptides recognizable by antibodies or capable of presentation by MHCmolecules, such as those at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 or >20 amino acids in length.

The term “polyclonal antibody” refers to antibodies that areheterogeneous populations of antibody molecules derived from the sera ofanimals immunized with an antigen or an antigenic functional derivativethereof. For the production of polyclonal antibodies, various hostanimals may be immunized by injection with the antigen. Variousadjuvants may be used to increase the immunological response, dependingon the host species.

The term “monospecific antibody” refers to an antibody or pool ofantibodies that recognize a specific epitope, antigenic determinant, orantigen. An epitope may be unique to MERS or other coronaviruses orshared between one or more viruses expressing N protein or N proteinanalogs. IgY antibodies may be specific for MERS or MERS-CoV N proteinor may cross-react with N proteins or N protein analogs of othercoronaviruses. In some embodiments monospecific antibodies are isolatedby affinity chromatography, for example, by binding to a substratecomprising an antigen, such as N protein, a domain or fragment of Nprotein, washing non-bound antibodies away, and elution of theantibodies bound to the antigen on the substrate. Monospecificantibodies may also be isolated or enriched by elution from a substrateto which they have bound such as a substrate comprising a peptideepitope of N protein.

The term “monoclonal antibody” is well recognized in the art and refersto an antibody that can be mass produced in the laboratory from a singleclone and that recognizes only one antigen or epitope. Monoclonalantibodies are typically made by fusing a normally short-lived,antibody-producing B cell to a fast-growing cell, such as a cancer cell(sometimes referred to as an “immortal” cell). The resulting hybridcell, or hybridoma, multiplies rapidly, creating a clone that produceslarge quantities of the antibody. “Monoclonal antibodies” aresubstantially homogenous populations of antibodies directed to aparticular antigen or epitope. They may be obtained by any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. Monoclonal antibodies may be obtained by methodsknown to those skilled in the art. See, for example, Kohler, et al.,Nature 256:495-497, 1975, and U.S. Pat. No. 4,376,110.

The term “crystalline” refers to an antibody, such as a monoclonalantibody that has been purified by crystallization, such as by batchcrystallization. Crystalline antibodies can be used in order to generatea small volume, highly concentrated forms. See Yang et al., 2003,Crystalline antibodies for subcutaneous delivery. PNAS,2003,100(12):6934-6939 which is incorporated by reference. The IgYantibodies disclosed herein may be crystallized.

The term “antibody fragment” encompasses any synthetic or geneticallyengineered protein that acts like an antibody by binding to a specificantigen to form a complex. For example, antibody fragments includeisolated fragments, “Fv” fragments, consisting of the variable regionsof the heavy and light chains, recombinant single chain polypeptidemolecules in which light and heavy chain variable regions are connectedby a peptide linker (“scFv proteins”), and minimal recognition unitsconsisting of the amino acid residues that mimic the hypervariableregion. Antibody fragments include a portion of an antibody such asF(ab′)2, F(ab)2, Fab′, Fab, Fv, sFv and the like. Regardless ofstructure, an antibody fragment binds with the same antigen that isrecognized by the intact antibody.

The term “specific binding” in the context of the characteristics ofspecific binding molecules, also known as specific targeted immunefactors, such as an antibody or antibody fragment, refers to the abilityto preferentially bind to a particular antigen that is present in ahomogeneous mixture of different antigens. In certain embodiments, aspecific binding interaction will discriminate between desirable andundesirable antigens (e.g., “target” and “non-target” antigens) in asample, in some embodiments more than about 10 to 100-fold or more(e.g., more than about 1000- or 10,000-fold). In some embodiments, thespecific binding molecule may specifically bind to an epitope sharedamong different species or strains of a virus like MERS-CoV as comparedto non-shared epitopes. In certain embodiments, the affinity between anantibody and antigen when they are specifically bound in anantibody-antigen complex is characterized by a K_(D) (dissociationconstant) of less than 10⁻⁶ M, less than 10 ⁻⁷ M, less than 10⁻⁸ M, lessthan 10⁻⁹ M, less than 10⁻¹⁰ M, less than 10⁻¹¹ M, or less than about10⁻¹² M or less.

Terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

It will be further understood that the terms “comprises” and/or“comprising,” when used in this specification, specify the presence ofstated features, steps, operations, elements, and/or components, but donot preclude the presence or addition of one or more other features,steps, operations, elements, components, and/or groups thereof.

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items and may be abbreviated as“/”.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “substantially”, “about” or“approximately,” even if the term does not expressly appear. The phrase“about” or “approximately” may be used when describing magnitude and/orposition to indicate that the value and/or position described is withina reasonable expected range of values and/or positions. For example, anumeric value may have a value that is +/− 0.1% of the stated value (orrange of values), +/− 1% of the stated value (or range of values), +/−2% of the stated value (or range of values), +/− 5% of the stated value(or range of values), +/− 10% of the stated value (or range of values),+/− 15% of the stated value (or range of values), +/− 20% of the statedvalue (or range of values), etc. Any numerical range recited herein isintended to include all sub-ranges subsumed therein.

Numerical ranges as used herein are intended to include every number andsubset of numbers contained within that range, whether specificallydisclosed or not. Further, these numerical ranges should be construed asproviding support for a claim directed to any number or subset ofnumbers in that range. For example, a disclosure of from 1 to 10 shouldbe construed as supporting a range of from 2 to 8, from 3 to 7, from 5to 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

Although the terms “first” and “second” may be used herein to describevarious features/elements (including steps), these features/elementsshould not be limited by these terms, unless the context indicatesotherwise. These terms may be used to distinguish one feature/elementfrom another feature/element. Thus, a first feature/element discussedbelow could be termed a second feature/element, and similarly, a secondfeature/element discussed below could be termed a first feature/elementwithout departing from the teachings of the present invention.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper”, “in front of” or “behind” and the like, may be used herein forease of description to describe one element or feature's relationship toanother element(s) or feature(s) as illustrated in the figures. It willbe understood that the spatially relative terms are intended toencompass different orientations of the device in use or operation inaddition to the orientation depicted in the figures. For example, if adevice in the figures is inverted, elements described as “under” or“beneath” other elements or features would then be oriented “over” theother elements or features. Thus, the exemplary term “under” canencompass both an orientation of over and under. The device may beotherwise oriented (rotated 90 degrees or at other orientations) and thespatially relative descriptors used herein interpreted accordingly.Similarly, the terms “upwardly”, “downwardly”, “vertical”, “horizontal”and the like are used herein for the purpose of explanation only unlessspecifically indicated otherwise.

When a feature or element is herein referred to as being “on” anotherfeature or element, it can be directly on the other feature or elementor intervening features and/or elements may also be present. Incontrast, when a feature or element is referred to as being “directlyon” another feature or element, there are no intervening features orelements present. It will also be understood that, when a feature orelement is referred to as being “connected”, “attached” or “coupled” toanother feature or element, it can be directly connected, attached orcoupled to the other feature or element or intervening features orelements may be present. In contrast, when a feature or element isreferred to as being “directly connected”, “directly attached” or“directly coupled” to another feature or element, there are nointervening features or elements present. Although described or shownwith respect to one embodiment, the features and elements so describedor shown can apply to other embodiments. It will also be appreciated bythose of skill in the art that references to a structure or feature thatis disposed “adjacent” another feature may have portions that overlap orunderlie the adjacent feature.

The description and specific examples, while indicating embodiments ofthe technology, are intended for purposes of illustration only and arenot intended to limit the scope of the technology. Moreover, recitationof multiple embodiments having stated features is not intended toexclude other embodiments having additional features, or otherembodiments incorporating different combinations of the stated features.Specific examples are provided for illustrative purposes of how to makeand use the compositions and methods of this technology and, unlessexplicitly stated otherwise, are not intended to be a representationthat given embodiments of this technology have, or have not, been madeor tested.

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference,especially referenced is disclosure appearing in the same sentence,paragraph, page or section of the specification in which theincorporation by reference appears.

The citation of references herein does not constitute an admission thatthose references are prior art or have any relevance to thepatentability of the technology disclosed herein. Any discussion of thecontent of references cited is intended merely to provide a generalsummary of assertions made by the authors of the references, and doesnot constitute an admission as to the accuracy of the content of suchreferences.

1. A method for detecting MERS-CoV in a sample, comprising contactingthe sample with a monospecific IgY antibody that recognizes MERS-CoVnucleocapsid protein (“N protein”) and detecting complex formation orbinding of the IgY antibody to the MERS-CoV.
 2. The method of claim 1,which is an ELISA sandwich assay, wherein the IgY antibody thatrecognizes MERS-CoV N protein is a capturing antibody bound to asubstrate, wherein the capturing antibody bound to the substrate iscontacted with the sample suspected of containing MERS-CoV, and whereincomplex formation is measured by the binding of a tagged secondaryantibody that recognizes MERS-CoV.
 3. The method of claim 1 that is animmunoswab assay, wherein said contacting comprises contacting thesample with IgY antibodies bound to a swab, removing unbound sample, anddetecting complex formation.
 4. The method of claim 1, wherein saidsample contains no more than 20 ng/ml of MERS-CoV N protein.
 5. Themethod of claim 1, wherein the IgY antibody does not substantiallyinteract with rheumatoid factor (RF), human anti-mouse IgG antibodies(HAMA), complement components and/or mammalian Fc receptors.
 6. Themethod of claim 1, wherein the IgY antibody is a polyclonal antibodyproduced by immunizing chickens with isolated or recombinant MERS-CoV Nprotein and recovering IgY antibody from eggs laid by the immunizedchickens.
 7. The method of claim 1, wherein the IgY antibody comprises apolyclonal monospecific IgY antibody for MERS-CoV N protein.
 8. Themethod of claim 1, wherein the IgY antibody comprises a monospecificantibody that binds to a peptide epitope of MERS-CoV N protein thatranges in length from 6 to 20 amino acids of the amino acid sequencedescribed by SEQ ID NO:
 1. 9. The method of claim 1, wherein the IgYantibody comprises a monospecific antibody that binds to an N-terminaldomain segment of MERS CoV N protein comprising residues 1-36 of SEQ IDNO:
 1. 10. The method of claim 1, wherein the IgY antibody comprises amonospecific antibody that binds to an N-terminal domain segment of MERSCoV N protein comprising residues 37-164 of SEQ ID NO:
 1. 11. The methodof claim 1, wherein the IgY antibody comprises a monospecific antibodythat binds to a central segment of MERS CoV N protein comprisingresidues 165-238 of SEQ ID NO: 1; or that binds to a disordered regionor LKR containing a stretch of serine and arginine residues andcomprising residues 156 to 206 of SEQ ID NO: 1 or (SEQ ID NO: 3)LPKNFHIEGTGGNSQSSSRASSVSRNSSRSSSQGSRSGNSTRGTSPGPSG I.


12. The method of claim 1, wherein the IgY antibody comprises amonospecific antibody that binds to a C-terminal domain segment of MERSCoV N protein comprising residues 239-362 of SEQ ID NO:
 1. 13. Themethod of claim 1, wherein the IgY antibody comprises a monospecificantibody that binds to a C-terminal domain segment of MERS CoV N proteincomprising residues 363-413 of SEQ ID NO:
 1. 14. The method of claim 1,wherein the IgY antibody comprises a monospecific antibody that binds toone or more residues in a peptide segment of SEQ ID NO: 1 selected fromthe group comprising g residues 4-12, 42-47, 50-58, 60-66, 92-102,108-126, 144-150, 172-180, 206-225, 230-240, 261-268, 296-304, 324-337,347-353and 399-405 described by SEQ ID NO:
 1. 15. The method of claim 1,wherein the IgY antibody comprises a monospecific antibody that binds toone or more residues in a peptide segment of SEQ ID NO: 1 selected fromthe group comprising: K346, W347, L348, and E349; Y327, F328, L329, andR330; L350, L351, E352, O353, N354, 1355, D356, A357, Y358, K359, T360,F361, P362, K363, K364, and E365; D320, D321, H322, G323, N324, P325,and V326; H252, K253, R254, T255, S256, T257, K258, S259, F260, N261 ,M262, V263, Q264, A265, F266, G267, L268, R269, G270, P271, G272, D273,L274, Q275, G276, N277, F278, G279, D280, L281, Q282, L283, N284, K285,L286, G287, T288, E289, D290, P291, and R292; R399, P400, S401, V402,Q403, P404, G405, P406, M407, 1408, D409, V410, N411, T412, and D413;W293, P294, 0295, 1296, A297, and E298; F312, K313, L314, T315, H316,Q317, N318, and N319; and L299, A300, P301, T302, A303, 5304, A305,F306, M307, G308, M309, S310, and Q311.
 16. The method of claim 1,wherein the IgY antibody comprises a mixture of two or more monospecificIgY antibodies to MERS CoV N protein epitopes or domains.
 17. Anantibody conjugate comprising a monospecific polyclonal IgY antibodythat binds to MERS-CoV N protein that is conjugated to at least onedetectable marker or to a therapeutic agent.
 18. The antibody conjugateof claim 17 that binds to the N terminal domain segment MERS-CoV Nprotein comprising residues 37-164 of SEQ ID NO:
 1. 19. The antibodyconjugate of claim 17 comprising a monospecific polyclonal IgY antibodythat binds to the C terminal domain segment of MERS-CoV comprisingresidues 239-362 of SEQ ID NO:
 1. 20. A method for treating a subject atrisk of being infected by, or infected by, MERS-CoV comprisingadministering an IgY antibody that binds to MERS-CoV N protein to thesubject.